Many higher animals have five different classes of immunoglobulins, IgG, IgA, IgM, IgD, and IgE. Each immunoglobulin class differs in properties such as size, charge, amino acid composition, and sugar content. Of these classes, IgM accounts for approximately 10% of all plasma immunoglobulins. IgM is the major component of early antibodies produced against cell-membrane antigens, infectious microorganisms, or soluble antigens, which have a complex antigenicity.
Human IgMs usually have a pentameric structure. Each of the five subunits constituting this pentameric structure has a four-stranded structure similar to that of IgG. The amino acid sequence of the μ chain, which is the heavy chain of IgM, is different from that of the γ chain, which is the heavy chain of IgG. The following differences can also be seen:                The μ chain has an extra constant domain than the γ chain.        The μ chain has four more oligosaccharide chains than the γ chain.        
IgM has a polypeptide chain called the J chain, which is not found in IgG. The J chain is considered to assist the association of μ chains prior to secretion of IgM from antibody producing cells.
With advances in monoclonal antibody technology and recombinant DNA technology, large-scale production of pure immunoglobulins has become possible in recent years. Furthermore, gene recombination techniques have enabled production of chimeric antibodies and humanized antibodies. Chimeric antibodies are antibodies having a structure in which the variable regions have been replaced with variable regions derived from a different species. For example, “chimeric antibodies” comprising variable regions of non-human antibodies and the constant regions of human antibodies (Non-Patent Document 1/Proc. Natl. Acad. Sci. U.S.A., (1984) 81:6851) are known. Also known are humanized antibodies in which the complementarity determining regions (CDR) of other animal species are transferred into human immunoglobulins (Non-Patent Document 2/Nature (1986) 321:522-525)
Actual examples of antitumor antibodies are the anti-CD20 human chimeric antibody Rituxan (IDEC), and the anti-HER2/neu humanized antibody Herceptin (Genentech), which have completed clinical trials and have already been approved. These antibodies are now commercially available. Antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC) activity and complement-dependent cytotoxicity (hereinafter referred to as CDC) activity are known as effector functions of IgG and IgM. Since IgM has a higher CDC activity compared to IgG, it has an extremely high chance of becoming an anti-tumor antibody having CDC activity as its main effect. However, as described above, unlike IgG, IgM forms a multimer. Therefore, industrial scale production of recombinant IgM had been considered difficult.
IgM is also very unstable compared to IgG and has a low solubility. Therefore, the production of a highly concentrated and stable IgM solution is difficult. For example, Cytotherapy, 2001, 3(3), 233-242 (Non-Patent Document 5) reports that, even when IgM had been stored at −20° C., precipitation and decrease of activity occurred upon thawing. Furthermore, according to the report, IgM easily aggregates and precipitates during storage. Arch. Pathol. Lab. Med., 1999, 123, 119-125 (Non-Patent Document 6) showed that among precipitates called cryoprecipitations or low-temperature precipitations observed in human serum, Type I cryoglobulin, which produces a precipitate consisting of a single antibody component, is mainly IgM. IgM, in particular, readily undergoes cryoprecipitation, making it difficult to obtain a highly concentrated IgM solution at a low temperature. Most biopharmaceuticals are stored and distributed under refrigeration at around 4° C. to ensure stability. Since some IgMs cryoprecipitate at around 4° C., it is preferable that their cryoprecipitation is suppressed during drug formulation, storage, and distribution. Cryoprecipitation also occurs in IgM bulk drug substance production processes leading to formulation, during purification and concentration steps at low temperature, and during low-temperature storage between the multiple steps involved. This causes operational problems, and thus, it is preferable to suppress cryoprecipitation even in these circumstances.
Various attempts have been made to stabilize IgM at low temperature. For example, Immunochemistry, 1978, 15, 171-187 (Non-Patent Document 3) discloses that cryoprecipitation of IgM takes place more readily with temperature decrease and concentration increase. It also discloses that cryoprecipitation takes place in the pH range of 5 to 10, and that this cryoprecipitation can be avoided at extremely high pH or low pH. However, antibodies generally tend to undergo a deamidation reaction and aggregation at high pH, and denaturation and aggregation at low pH. Antibodies are generally known to be chemically and physically stable from pH5 to pH8, especially near pH5 to pH7. It is therefore difficult to ensure a stability sufficient enough to withstand pharmaceutical use at extremely high pH or low pH.
Journal of Biological Chemistry, 1977, 252(22), 8002-8006 (Non-Patent Document 4) examined the effect of various compounds on cryoprecipitation (solubility of IgM at low temperature), and discloses that cryoprecipitation decreases when sugars are added or salt concentration is increased. However, this disclosure shows that for effective prevention of cryoprecipitation using any sugars or salts, the sugars or salts must be added at high concentrations of approximately 500 mM or higher. When used as a pharmaceutical, it is preferable to achieve such an effect at lower concentrations.
WO 91/18106 (Patent Document 1) discloses methods for preventing cryoprecipitation by changing the structure of sugar chains attached to IgM. However, when sugar chains of antibodies are modified, in some cases, the binding activities of antibodies change. Therefore, it is desirable to develop methods for suppressing cryoprecipitation without altering the structure of antibodies, including their sugar chains.
Patent Document 1: WO 91/18106
Non-Patent Document 1: Proc. Natl. Acad. Sci. U.S.A, (1984) 81: 6851
Non-Patent Document 2: Nature (1986) 321: 522-525
Non-Patent Document 3: Immunochemistry, 1978, 15, 171-187
Non-Patent Document 4: Journal of Biological Chemistry, 1977, 252(22), 8002-8006
Non-Patent Document 5: Cytotherapy, 2001, 3(3), 233-242
Non-Patent Document 6: Arch. Pathol. Lab. Med., 1999, 123, 119-125